Autophagy promotes non-lytic release of progeny rhinovirus in microvesicles from mucociliary differentiated airway epithelial cells

Abstract

Airway epithelial cell cultures from patients with chronic obstructive pulmonary disease (COPD) show higher viral loads following rhinovirus (RV) infection in both extracellular and intracellular compartments, despite expressing antiviral interferon responses. Because autophagy is implicated in viral replication and is elevated in COPD airway epithelial cells, we investigated its role in enhancing viral load in these cells. Mucociliary-differentiated airway epithelial cell cultures from healthy non-smokers and COPD patients were infected apically with RV. In selected experiments, COPD cultures were treated with the autophagy inhibitor 3-methyladenine (3-MA). Apical washes were collected, microvesicles were isolated, and viral load was quantified by plaque assay and qPCR. BEAS-2B cells were transfected with ICAM-1, LC3β or control siRNA and infected with either naked RV or microvesicle-encapsulated RV to assess viral RNA levels and interferon responses. COPD airway epithelial cultures released significantly more microvesicles enriched with infectious progeny virus than normal cultures. RV infection increased autophagy in both normal and COPD. Inhibition of autophagy reduced the release of microvesicle-associated virus without altering total viral load. Knockdown of ICAM-1 significantly reduced infection by naked virus but had no effect on infection by microvesicle-encapsulated virus. These results indicate the contribution of autophagy to the release of macrovesicles-encapsulated progeny virions without causing cell death. RV-infected COPD cells release more progeny virions in microvesicles, enabling receptor-independent infection of neighboring cells which may contribute to sustained viral burden despite antiviral responses.